Pyrosequencing in a miniaturized device has been shown to have wide applications in genome sequencing. However, pyrosequencing with natural nucleotides has inherent difficulty in accurately deciphering the homopolymeric regions of DNA templates. The goal of this proposal is to design and synthesize a library of reversible nucleotide terminators to address this issue. The following three aims will be pursued: (1) Design and synthesis of four 3'-O-allyl-labeled nucleotides corresponding to A, C, G, T, as reversible terminators for pyrosequencing. We have produced small quantity of four 3'-O-allyl-labeled nucleotides in sufficient purity and have demonstrated that they can be used for pyrosequencing to produce accurate sequencing data in homopolymeric regions of DNA. We will further optimize the enzymatic conditions to increase the read length of pyrosequencing using the 3'-O-allyl-labeled nucleotides; (2) Design and synthesis of photocleavable 3'-O-modified nucleotide reversible terminators to compare with the 3'-O-allyl-labeled nucleotides to further optimize the pyrosequencing platform in terms of accuracy and readlength; (3) Evaluation of different micron-sized beads to immobilize the DNA template that are compatible to photocleavage or chemical cleavage conditions to perform pyrosequencing using the optimized set of reversible nucleotide terminators developed above. The molecular tools developed here will facilitate the optimization of pyrosequencing for de novo genome sequencing with unparalleled accuracy for biological and biomedical applications. [unreadable] [unreadable] [unreadable] [unreadable]